Fluorescence is the process of monitoring fluorescent radiation from an object for analysis, characterization or imaging. Typically, an excitation pulse of radiation is directed onto or into a sample, followed by fluorescence of the sample, and the detection of the fluorescent radiation. The detected fluorescence is used for sample analysis, characterization or imaging. In the case of an immuneassay, analysis of a sample is typically done by marking a desired species with a fluorescable tag, exciting the sample and monitoring for fluorescence from the tag.
Theoretically, fluorometry is capable of being the most sensitive of all analytic tools. It is possible to detect single photon events, and possible to re-excite a fluorophore and confirm the analysis. However, the problem which has plagued fluorescence has been in discriminating the fluorescent signal of interest from the background radiation in the system. Often times, the signal from "background" radiation may be 10,000 times larger than the intensity of the fluorescent signal of interest. Detection of the unwanted background radiation reduces the image quality and accuracy of the detection.
The problem caused by background radiation is particularly acute in biological systems. For example, in the analysis of blood plasma, the presence of a naturally occurring fluorescable material, such as biliverdin, causes substantial background radiation. Other sources of undesirable background radiation include ambient radiation, radiation from fast fluorescing materials (generally considered to be those with decay half lives on the order of 1 to 1.5 nanoseconds) and various scattering mechanisms, such as Raman scattering bands.
Earlier attempts to overcome the problem of background radiation have met with limited success. A first technique involves discriminating against background radiation on the basis of wavelength. Generally, a filter is used to reject detected radiation at all but a narrowly defined wavelength band. This technique has been less than successful principally because the background radiation may also be at the same wavelength as the desired fluorescence signal, and accordingly, still be passed through the filter and detected.
A second technique attempting to discriminate the desired fluorescent signal from the background is the so called time gating approach. Here, the fluorescent signal is observed in a short time window after the excitation. The time window may be varied both in its length and in its starting time. Through the use of the variable time window, the detected radiation may be observed at the maximal time for detection sensitivity- Historically, this technique has used a fluorophore of very long decay time (such as 1,000 nanoseconds) to allow the background fluorescence to substantially decay before detection of the fluorescent signal of interest. Generally however, long decay time fluorophores are less desirable than shorter decay time fluorophores because they are relatively insensitive and require longer times for overall analysis.
Historically, there have been two excitation pulse formats for transient state fluorescent analysis. One format utilizes a single, relatively high power pulse which excites the fluorophore. The transient state is typically monitored by a high speed photomultiplier tube by monitoring the analog signal representative of current as a function of time. Single pulse systems require sufficiently high power to excite a large number of fluorescent molecules to make detection reliable. The other principal format for transient state fluorescent analysis utilizes repetitive excitation pulses. Ordinarily, pulses of relatively short, typically nanosecond duration, light with power in the microwatt range are repetitively supplied to the sample at rates varying from 1 to 10,000 Hz. Ordinarily, the excitation source is a lamp, such as a Xenon-lamp. Frequently, the decay curve is measured digitally by determining the time to receipt of a photon. One commercially available system uses repetitive pulses (such as at 5,000 Hz) and strobes the photomultiplier tube at increasingly longer times after the flash in order to obtain a time dependent intensity signal. Detection in such systems has proved to be very time consuming and insensitive. A single analysis can take on the order of one hour, even at relatively high fluorescable dye concentrations (e.g. 10.sup.-6 M).
Recently, significant advances have been made in the area of fluorescable dyes. In one aspect, dyes being excitable by longer wavelength radiation, such as in the red and infrared wavelengths, are now available. Applicant incorporates by reference the applications by Arrhenius, U.S. patent application Ser. No. 701,449, filed May 15, 1991, entitled, "Fluorescent Marker Components and Fluorescent Probes," (which is a continuation-in-part of U.S. patent application Ser. No. 523,601, filed May 15, 1990), and Dandliker and Hsu, U.S. patent application Ser. No. 701,465, filed May 15, 1991 entitled "Fluorescent Dyes Free of Aggregation and Serum Binding" (which is a continuation-in-part of U.S. patent application Ser. No. 524,212, filed May 15, 1990). Significant improvements in sensitivity are achieved through use of these modern dyes over older dyes.
Further significant advancements have been made in increasing sensitivity through data collection and analysis techniques. As disclosed in Dandliker et al., U.S. Pat. No. 4,877,965, entitled "Fluorometer," time gating techniques are used in conjunction with data collection and analysis techniques to obtain an improved signal relative to the background. Generally, Dandliker et al., considers the detected intensity as a function of time to be composed of signals from various sources, including the desired signal source, and various undesired background sources. Optimization of the desired signal is achieved through data collection and analysis techniques.
Further significant advancements have been made in the ability to measure relevant materials in immunoassays. For example, in Dandliker et al, U.S. patent application Ser. No. 490,770, filed Mar. 6, 1990, entitled "Transient State Luminescence Assays," (which is a continuation-in-part of U.S. patent application Ser. No. 365,420, filed Jun. 13, 1989) incorporated herein by this reference, the bound and free form of materials in a homogeneous assay may be determined. Generally, the technique requires measurement of the time dependent decay of the intensity of parallel and perpendicular polarization components. By measuring the time dependent decay of various polarization states, it is possible to determine the bound and free forms of materials such as haptens, peptides, or small proteins in a homogeneous analysis format. Significantly, no separation of the bound and free materials is required.
Despite the significant and promising improvements made in the field of fluorescable dyes, and in the data analysis aspects, the actual methods and apparatus for achieving and detecting fluorescence have heretofore remained relatively unchanged. Utilizing even the most sensitive and best equipment, analysis can take an hour or more, even at high concentrations of materials. When fluorometry is used for immunoassay in a clinical context, time for analysis and proper diagnosis can be absolutely critical. Patient survival can depend on accurate, timely analysis. Additionally, rapid testing would permit retests of patients without having them wait significant periods of time, resulting in more rapid and accurate diagnosis. As to sensitivity, it is extremely desirable to be able to detect minute amounts of fluorescable material. However, as the amount of fluorescable material in a sample decreases, the ratio of the size of the undesired background signal to the desired signal increases. Further, since the time for analysis depends on the amount of fluorescent radiation received from the detector, low concentrations generally require substantially more time to analyze.
Heretofore, the time required for analysis has been prohibitively high. Known methods and apparatus have failed to provide rapid and accurate diagnosis and analysis of samples. This has been so despite the clear and known desirability of the use of fluorometry.